CROSS REFERENCE TO RELATED APPLICATIONS
A commonly owned application, U.S. Ser. No. 08/322,137, filed Oct. 13, 1994, incorporated by reference herein, relates to use of engineered yeast cells in screening for substances which modulate the activity of a mammalian surrogate of a yeast pheromone system protein, e.g., the yeast pheromone receptor, a G protein-coupled receptor.
1. Field of the Invention
The invention relates inter alia, to expression of a mammalian adenylyl cyclase in yeast, the transformed yeast cells, and their use, e.g., in identifying potential inhibitors or activators of the mammalian adenylyl cyclase, or of other proteins which are natively or artificially coupled to the mammalian adenylyl cyclase in the engineered yeast cell.
2. Description of the Background Art
Signal Transduction
In some instances, for a drug to cure a disease or alleviate its symptoms, the drug must be delivered to the appropriate cells, and trigger the proper "switches." The cellular switches are known as "receptors." Hormones, growth factors, neurotransmitters and many other biomolecules normally act through interaction with specific cellular receptors. Drugs may activate or block particular receptors to achieve a desired pharmaceutical effect. Cell surface receptors mediate the transduction of an "external" signal (the binding of a ligand to the receptor) into an "internal" signal (the modulation of a pathway in the cytoplasm or nucleus involved in the growth, metabolism or apotosis of the cell).
In many cases, transduction is accomplished by the following signaling cascade:
An agonist (the ligand) binds to a specific protein (the receptor) on the cell surface. PA1 As a result of the ligand binding, the receptor undergoes an allosteric change which activates a transducing protein in the cell membrane. PA1 The transducing protein activates, within the cell, production of so-called "second messenger molecules." PA1 The second messenger molecules activate certain regulatory proteins within the cell that have the potential to "switch on" or "off" specific genes or alter some metabolic process.
This series of events is coupled in a specific fashion for each possible cellular response. The response to a specific ligand may depend upon which receptor a cell expresses. For instance, the response to adrenalin in cells expressing .alpha.-adrenergic receptors may be the opposite of the response in cells expressing .beta.-adrenergic receptors.
The above "cascade" is idealized, and variations on this theme occur. For example, a receptor may act as its own transducing protein, or a transducing protein may act directly on an intracellular target without mediation by a "second messenger".
Signal Transduction Through G Proteins
Signals initiated by a variety of mammalian hormones and neurotransmitters are received by seven transmembrane domain receptors in the plasma membrane of cells and are transduced to intracellular effectors via heterotrimeric G proteins. Many different G proteins are known to interact with receptors. G protein signaling systems include three components: the receptor itself, a GTP-binding protein (G protein), and an intracellular target usually a protein.
The cell membrane acts as a switchboard. Messages arriving through different receptors can produce a single effect if the receptors acts on the same type of G protein. On the other hand, signals activating a single receptor can produce more than one effect if the receptor acts on different kinds of G proteins, or if the G proteins can act on different effectors.
The heterotrimeric G protein is composed of a guanine nucleotide-binding a subunit together with a tight complex of .beta. and .gamma. subunits. In their resting state, the G proteins, which consist of alpha (.alpha.), beta (.beta.) and gamma (.gamma.) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with receptors. When a hormone or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This spurs the a subunit to release GDP, and the more abundant nucleotide guanosine tri-phosphate (GTP), replaces it, activating the G protein. The G protein then dissociates to separate the .alpha. subunit from the still complexed beta and gamma subunits. The free G.alpha. and the G.beta..gamma. subunits both may be capable of influencing the activity of specific effector molecules (e.g., the enzymes adenylyl cyclase, cyclic GMP phosphodiesterase (PDE), phospholipase C, phospholipase A.sub.2, and selected ion channels). The effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active "second messenger," which may diffuse through the cytoplasm, triggering a metabolic cascade. After a few seconds, G protein signalling is terminated with the hydrolysis of GTP to GDP through the intrinsic GTPase activity of the G.alpha. subunit and the subsequent reassociation of G.alpha.-GDP with G.beta..gamma. to form the inactive heterotrimer. This reassociation is driven by the high affinity of GDP-bound G.alpha. for G.beta..gamma..
Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Although the greatest variability has been seen in the a subunit, several different .beta. and .gamma. structures have been reported. There are, additionally, several different G protein-dependent effectors.
The study of microorganisms indicates that the development of G protein signal transduction pathways arose early in the evolution of eukaryotic cells. G protein regulatory function is intrinsic to the response to mating pheromones in yeast (Whiteway et al. 1989) and the development of the cellular slime mold Dictyostelium discoideum is controlled by G protein-mediated responses to cAMP (Devreotes 1989).
The Role of Adenylyl Cyclase in G-Protein-Mediated Signal Transduction
Adenylyl cyclase is among the best studied of the effector molecules which function in mammalian cells in response to activated G proteins. Activation of adenylyl cyclase occurs when signals transduced from specific cellular receptors result in the release of GTP-bound G.alpha.s. G.alpha.s ("s" denotes stimulatory) was originally identified as a regulator of adenylyl cyclase activity in mutant S49 cells which lacked adenylyl cyclase activity. G.alpha.s-GTP stimulated adenylyl cyclase activity in those cyc.sup.- cells (Northup et al. (1980) Proc. Natl. Acad. Sci. USA 77, 6516-6520). The production of cAMP can be stimulated by pure GTP-.gamma.S-bound G.alpha.s (GTP-.gamma.S is a non-hydrolyzable form of the nucleotide). Activation of cyclase by GTP-bound G.alpha.s is reversed by excess G.beta..gamma.; inhibition is assumed to occur as an inactive G protein heterotrimer re-forms.
Molecules which signal through receptors that interact with another class of G proteins, G.alpha.i (including G.alpha.i1, G.alpha.i2, and G.alpha.i3), mediate inhibition of adenylyl cyclase. Upon agonist binding to Gi-coupled receptors, both activated G.alpha.i protein and the released G.beta..gamma. complex appear to be capable of inhibiting the activity of adenylyl cyclase [Taussig et al. (1993) Science 261, 218-221]. The G.beta..gamma. complex may inhibit the enzyme's activity by reforming a heterotrimer with free G.alpha.s, thereby sequestering that stimulatory molecule (Gilman (1984) Cell 36, 577-579). In addition, the G.alpha.i subunit may directly inhibit adenylyl cyclase activity (Taussig et al. (1993) Science 261, 218-221.) A third mechanism for the negative regulation of adenylyl cyclase involves direct inhibition by the G.beta..gamma. complex. Purified type 1 adenylyl cyclase has been shown to be directly inhibited by .beta..gamma. subunits (Taussig et al. (1993) J. Biol. Chem. 268, 9-12).
Cyclic nucleotides play an important role in the regulation of a multitude of cellular activities. The synthesis of adenosine 3', 5'-cyclic phosphate (cyclic adenosine monophosphate or cAMP) is catalyzed by adenylyl cyclase, an enzyme which, in mammalian cells, is an integral membrane protein. Cyclic AMP is a second messenger which acts in response to cellular signals through a specific protein kinase (cAMP-dependent protein kinase or protein kinase A) to phosphorylate target molecules, e.g., other protein kinases or proteins involved in transport or cellular morphology. Through stimulation of the kinase, intracellular cAMP mediates many of the effects of hormones in the regulation of cellular metabolism and cell growth. Cyclic AMP is hydrolyzed by several phosphodiesterases (PDE) and can be actively secreted from some cell types, presumably via a specific transporter, or sequestered from the cytoplasm via transporters present in the membranes of intracellular organelles.
In vertebrate cells, adenylyl cyclase is regulated by heterotrimeric G proteins [Gilman (1984) Cell 36, 577-579] while in yeast, RAS proteins regulate adenylyl cyclase [Toda et al. (1985) Cell 40, 27-36; Broek et al. (1985) Cell 41, 763-769]. In turn, the activity of both the heterotrimeric G proteins and RAS proteins are controlled by the forms of guanine nucleotides to which they are bound.
While most adenylyl cyclases are found associated with the plasma membrane, certain forms of the enzyme expressed in bacteria are cytosolic, as is a mammalian enzyme found in testis. Peripheral membrane adenylyl cyclases are expressed in E. coli (Aiba et al. 1984) and in S. cerevisiae (Kataoka et al. 1985). The adenylyl cyclase encoded by the ACG gene of Dictyostelium appears to have a single transmembrane domain (Pitt et al. 1992). A second adenylyl cyclase gene from Dictyostelium (ACA) (Pitt et al. 1992), the Drosophila rutabaga gene (Levin et al. 1992), and the six full-length cDNAs encoding mammalian adenylyl cyclases that have been cloned to date code for integral membrane proteins.
Yeast Pheromone System Proteins and Their Metabolic Function
Haploid yeast cells are able not only to grow vegetatively, but also to mate to form a diploid cell. The two mating types ("sexes") of haploid cells are designated a and .alpha.. The a cells produce the dodecapeptide a-factor, and the .alpha. cells, the tridecapeptide .alpha.-factor. Because a-factor and .alpha.-factor elicit a mating response in the yeast cell of the opposite "sex", they are called "pheromones". These pheromones, as well as other proteins specifically involved in the production or transport of, or response to, pheromones, are considered "pheromone system proteins".
The gene encoding a-factor pheromone, like the .alpha.-factor receptor gene, is an a cell-specific gene; a cell-specific genes are only expressed in a cells. The gene encoding .alpha.-factor pheromone, like the a-factor receptor gene, is an .alpha. cell-specific gene; .alpha. cell-specific genes are only expressed in a cells. Other yeast genes belong to a haploid-specific gene set and are expressed in haploid cells (a cells or .alpha. cells) but not in diploid (a/.alpha.) cells. In addition, there exists a diploid cell-specific gene set, including those genes involved in sporulation.
In eukaryotic cells, RNA polymerase II promoters contain a specific sequence (the TATA box) to which the transcription factor TFIID (TATA binding protein or TBP) binds. An active transcription initiation complex includes TFIID, accessory initiation proteins, and RNA Pol II. As in higher eukaryotic cells, the TATA box is an essential control sequence in yeast promoters. Yeast TATA-box-binding protein (TBP) was identified by its ability to substitute in function for mammalian TFIID [Buratowski et al., Nature 334, 37 (1988); Cavallini et al., Nature 334, 77 (1988)]. With only a few apparent exceptions [transcription of some glycolytic enzyme genes, see Struhl, Mol. Cell. Biol. 6, 3847 (1986) and Ogden et al., Mol. Cell Biol. 6, 4335 (1986)] transcription of yeast genes requires the proximal TATA box element and TFIID binding for initiation of transcription. Also required for efficient transcription are gene-specific activator proteins; the precise mechanism whereby these gene-specific regulatory proteins influence transcription has not been completely elucidated.
MCM1p (encoded in the MCM1 gene) is a non-cell-type-specific transcription factor in yeast. MCM1p acts alone or in concert with other regulatory proteins to control expression of a- and .alpha.-cell specific genes. Yeast mating type loci encode the regulatory proteins that contribute to the control of cell type-specific expression. These proteins are Mata1p (encoded by the MATa gene) and Mat.alpha.1p and Mat.alpha.2p (encoded by the MAT.alpha. locus). MCM1p activates transcription of a-specific genes by binding to an upstream activation sequence (UAS) located in the control region of a-specific genes. Mat.alpha.1p and MCM1p interact to enhance each other's binding to specific UAS binding sites to activate .alpha.-cell-specific gene transcription in .alpha.-cells. Mat.alpha.2p associates with MCM1p to repress a-specific gene transcription in .alpha.-cells. In diploid (a/.alpha.) cells, Mat.alpha.1p and Mat.alpha.2p associate to repress the transcription of haploid-specific genes. The Mat.alpha.1p/Mat.alpha.2p regulatory entity is found only in diploid cells.
Yeast contain two genes encoding the .alpha.-factor pheromone, MF.alpha.1 and MF.alpha.2. Analysis of yeast bearing mutations in these sequences indicates that MF.alpha.1 gives rise to the majority of .alpha.-factor produced by cells. Expression occurs at a higher level from MF.alpha.1 than from MF.alpha.2 (Kurjan, Mol. Cell. Biol. 5, 787 (1985). The MF.alpha.1 gene of yeast encodes a 165 aa precursor protein containing an 85 aa leader sequence at the N-terminus. The leader includes a 19 aa signal sequence and a 66 aa sequence which contains sites for the addition of three oligosaccharide side chains (Kurjan and Herskowitz, Cell 39, 933 (1982); Singh et al. Nuc. Acids Res. 11, 4049 (1983); Julius et al. Cell 36, 309 (1984). Four tandem copies of the 13 aa .alpha.-factor are present in the C-terminal portion of the precursor; 6-8 aa spacer peptides precede the .alpha.-factor sequences (see FIG. 2).
After translocation of the nascent .alpha.-factor polypeptide to the ER, the signal sequence is cleaved from the precursor protein to yield pro-.alpha.-factor (Waters et al. J. Biol. Chem. 263, 6209 (1988). The core N-linked carbohydrate is added to three sites in the N-terminus of pro-.alpha.-factor (Emter et al. Biochem. Biophys. Res. Commun. 116, 822 (1983); Julius et al. Cell 36, 309 (1984); Julius et al. Cell 37, 1075 (1984). Additional glycosylation occurs in the Golgi prior to cleavage of pro-.alpha.-factor by the KEX2 endopeptidase. This enzyme cleaves within each of the spacer repeats leaving a Lys-Arg sequence attached to the C-terminus of .alpha.-factor peptide (Julius et al. Cell 37, 1075 (1984). The Lys-Arg sequence is removed by the action of the KEX-1 protease (Dmochowska et al. Cell 50, 573 (1987). The additional spacer residues present at the N-terminus of .alpha.-factor peptide are removed by the dipeptidyl aminopeptidase encoded by STE13 (Julius et al. Cell 32, 839 (1983). Four .alpha.-factor peptides are released from each precursor protein via the proteolytic processing outlined above and the mature .alpha.-factor is secreted from the cell.
Precursors of the 12 aa mature a-factor peptide are encoded in the MFa1 and MFa2 genes and are 36 aa and 38 aa residues, respectively (for schematic of MFa1 gene see FIG. 5). The precursors contain one copy of a-factor and the products of the two genes differ in sequence at one amino acid. The two forms of a-factor are produced in equal amounts by a cells (Manney et al. in Sexual interactions in eukaryotic microbes, p21, Academic Press, New York (1981).
Processing of a-factor entails a process that differs in every detail from that of .alpha.-factor. The processing of a-factor begins in the cytosol and involves the farnesylation of the C-terminal cysteine residue near the carboxyl terminus (-CVIA) by a farnesyl transferase (Schafer et al. Science 245, 379 (1989); Schafer et al. Science 249, 1133 (1990). The .alpha. and .beta. subunits of the farnesyl transferase are encoded by the RAM2 and RAM1 genes, respectively (He et al. Proc. Natl. Acad. Sci. 88, 11373 (1991). Subsequent to farnesylation is the proteolytic removal of the three amino acids that are C-terminal to the modified cysteine by a membrane-bound endoprotease. Next, the carboxy-terminal farnesylated cysteine residue is modified further: the carboxyl group is methylated by the product of the STE14 gene. STE14p is a membrane-bound S-farnesyl-cysteine carboxyl methyl transferase (Hrycyna et al. EMBO. J. 10, 1699 (1991). The mechanisms of the N-terminal processing of a-factor have not been elucidated. After processing of the precursors is complete, mature a-factor is transported to the extracellular space by the product of the STE6 gene (Kuchler et. al. EMBO J. 8, 3973 (1989), an ATP-binding cassette (ABC) transporter.
In normal S. cerevisiae (budding yeast) a cells, the .alpha.-factor binds the G protein-coupled membrane receptor STE2. The G protein dissociates into the G.sub..alpha. and G.sub..beta..gamma. subunits, and the G.sub..beta..gamma. binds an unidentified effector, which in turn activates a number of genes. STE20, a kinase, activates STE5, a protein of unknown function. STE5 activates STE11 kinase, which stimulates STE7 kinase, which induces the KSS1 and/or FUS3 kinases. These switch on expression of the transcription factor STE12. STE12 stimulates expression of a wide variety of genes involved in mating, including FUS1 (cell fusion), FAR1 (cell-cycle arrest), STE2 (the receptor), MFA1 (the pheromone), SST2 (recovery), KAR3 (nuclear fusion) and STE6 (pheromone secretion). Other genes activated by the pathway are CHS1, AG.alpha.1, and KAR3. The multiply tandem sequence TGAAACA has been recognized as a "pheromone response element" found in the 5'-flanking regions of many of the genes of this pathway.
One of the responses to mating pheromone is the transient arrest of the yeast cell in the G1 phase of the cell cycle. This requires that all three G1 cyclins (CLN1, CLN2, CLN3) be inactivated. It is believed that FUS3 inactivates CLN3, and FAR1 inhibits CLN2. (The product responsible for inactivating CLN1 is unknown).
The growth arrest is terminated by a number of different mechanisms. First, the .alpha.-factor receptor is internalized following binding of the pheromone, resulting in a transient decrease in the number of pheromone binding sites. Second, the C-terminal tail of the receptor is phosphorylated consequent to ligand binding, resulting in uncoupling of the receptor from the transducing G proteins. Third, pheromone-induced increases in expression of GPA1p (the G.alpha.-subunit of the heterotrimeric G protein) increase the level of the .alpha. subunit relative to the G.sub..beta. and G.sub..gamma. subunits, resulting in reduction in the level of free G.sub..beta..gamma. and consequent inactivation of the pheromone response pathway. Additional mechanisms include induction of the expression of SST2 and BAR1 and phosphorylation of the .alpha. subunit (perhaps by SVG1).
Signaling is inhibited by expression of a number of genes, including CDC36, CDC39, CDC72, CDC73, and SRM1. Inactivation of these genes leads to activation of the signaling pathway.
A similar pheromone signaling pathway may be discerned in .alpha. cells, but the nomenclature is different in some cases (e.g., STE3 instead of STE2).
Other yeast also have G protein-mediated mating factor response pathways. For example, in the fission yeast S. pombe, the M factor binds the MAP3 receptor, or the P-factor the MAM2 receptor. The dissociation of the G protein activates a kinase cascade (BYR2, BYR1, SPK1), which in turn stimulates a transcription factor (STE11). However, in S. pombe, the G.alpha. subunit transmits the signal, and there are of course other differences in detail.
Expression of Foreign Proteins in Yeast Cells
A wide variety of foreign proteins have been produced in S. cerevisiae, that remain in the yeast cytoplasm or are directed through the yeast secretory pathway (Kingsman et al. TIBTECH 5, 53 (1987). These proteins include, as examples, insulin-like growth factor receptor (Steube et al. Eur. J. Biochem. 198, 651 (1991), influenza virus hemagglutinin (Jabbar et al. Proc. Natl. Acad. Sci. 82, 2019 (1985), rat liver cytochrome P-450 (Oeda et al. DNA 4, 203 (1985) and functional mammalian antibodies (Wood et al. Nature 314, 446 (1985). Use of the yeast secretory pathway is preferred since it increases the likelihood of achieving faithful folding, glycosylation and stability of the foreign protein. Thus, expression of heterologous proteins in yeast often involves fusion of the signal sequences encoded in the genes of yeast secretory proteins (e.g., .alpha.-factor pheromone or the SUC2 [invertase] gene) to the coding region of foreign protein genes.
A number of yeast expression vectors have been designed to permit the constitutive or regulated expression of foreign proteins. Constitutive promoters are derived from highly expressed genes such as those encoding glycolytic enzymes like phosphoglycerate kinase (PGK1) or alcohol dehydrogenase I (ADH1) and regulatable promoters have been derived from a number of genes including the galactokinase (GAL1) gene. In addition, supersecreting yeast mutants can be derived; these strains secrete mammalian proteins more efficiently and are used as "production" strains to generate large quantities of biologically active mammalian proteins in yeast (Moir and Davidow, Meth. in Enzymol. 194, 491 (1991).
Heterologous G protein-coupled receptors have been functionally expressed in S. cerevisiae. Marsh and Hershkowitz, (Cold Spring Harbor Symp., Quant. Biol., 53: 557-65 (1988)) replaced the S. cerevisiae STE2 with its homologue from S. Kluyven. More dramatically, a mammalian beta-adrenergic receptor and G.alpha. subunit have been expressed in yeast and found to control the yeast mating signal pathway. King, et al., Science, 250: 121-123 (1990).
Duke University, WO92/05244 (Apr. 2, 1992) describes a transformed yeast cell which is incapable of producing a yeast G protein .alpha. subunit, but which has been engineered to produce both a mammalian G protein .alpha. subunit and a mammalian receptor which is "coupled to" (i.e., interacts with) the aforementioned mammalian G protein .alpha. subunit. Specifically, Duke reports expression of the human beta-2 adrenergic receptor (h.beta.AR), a seven transmembrane receptor (STR), in yeast, under control of the GAL1 promoter, with the h.beta.AR gene modified by replacing the first 63 base pairs of coding sequence with 11 base pairs of noncoding and 42 base pairs of coding sequence from the STE2 gene. (STE2 encodes the yeast .alpha.-factor receptor). Duke co-expressed a rat G protein .alpha. subunit in the same cells, yeast strain 8C, which lack the cognate yeast protein. Duke found that the modified h.beta.AR was functionally integrated into the membrane, as shown by studies of the ability of isolated membranes to interact properly with various known agonists and antagonists of h.beta.AR. The ligand binding affinity for yeast-expressed h.beta.AR was said to be nearly identical to that observed for naturally produced h.beta.AR. Ligand binding resulted in G protein-mediated signal transduction. Duke did not co-express a mammalian adenylyl cyclase in these cells.
Expression of Heterologous Adenylyl Cyclases in Yeast
African trypanosomes are protozoan parasites which are able to evade host immune defenses by altering their surface glycoproteins. The variable antigenicity is accomplished by sequential expression of genes encoding coat proteins. The variable surface glycoprotein genes (VSG) are transposed from silent regions to active, telomere-linked expression sites. Additional open reading frames (ORFs) termed the Expression Site Associated Genes (ESAGs), are found at these expression sites. ESAG4, cloned from Trypanosoma brucei, contains a sequence which is homologous to S. cerevisiae adenylyl cyclase [Pays et al. (1989) Cell 57, 835-845]. In addition, an ESAG from Trypanosoma equiperdum (eESAG4c), which is homologous to the ESAG4 of T. brucei, has been shown to encode an adenylyl cyclase which will complement an adenylyl cyclase (cyr-1) deletion mutant of S. cerevisiae [Ross et al. (1991) EMBO J. 10, 2047-2053].
The eESAG4c ORF contains sequence with homology to both S. cerevisiae and S. pombe adenylyl cyclases [Kataoka et al. (1985) Cell, 43, 493-505; Yamawaki-Kataoka et al. (1989) PNAS 86, 5693-5697; Young et al. (1989) PNAS 86, 7989-7993]. The region that is conserved between trypanosomes and yeast is within the yeast adenylyl cyclase catalytic domain and exhibits a sequence identity on the order of 50%. The eESAG4c sequence is approximately 40% identical to that of bovine brain adenylyl cyclase type 1 (Krupinski et al. (1990) Science 244, 1558-1562). The protein predicted by the eESAG4c sequence bears an N-terminal sequence that encodes a putative transmembrane domain flanking the sequence that is homologous to the adenylyl cyclase catalytic domain.
Also identified within the ESAG of Trypanosoma equiperdumare sequences which bear homology to a "leucine-rich repeat" gene family (Takahashi et al. (1985) PNAS 82, 1906-1910; Lopez et al. (1988) PNAS 85, 2135-2139; Hashimoto et al. (1988) Cell 52, 269-279). Proteins encoded by members of this family are involved in diverse functions, however, the repeat sequences are believed to be involved in membrane association and in protein-protein interactions. In S. cerevisiae the repeat domain of adenylyl cyclase is required for regulation of the enzyme by RAS proteins and for the association of the enzyme with the plasma membrane [Colicelli et al. (1990) Mol. Cell. Biol. 10, 2539-2543; Field et al. (1990) Science 247, 464-467; Mitts et al. (1990) Mol. Cell. Biol. 10, 3873-3883.] Also within the ESAG are sequences with limited homology to nucleotide binding domains [Florent et al. (1991) Mol. Cell. Biol. 11, 2180-2188] that have been hypothesized to have a regulatory function in trypanosomes analogous to that of Ras in yeast. Neither the leucine-rich repeat region nor the nucleotide binding domain were included in the sequences that complemented the yeast cyr deletion mutants [Ross et al. (1991) EMBO J. 10, 2047-2053]. Ross et al. (1991) speculated that the lack of these potential regulatory sequences would account for the much greater adenylyl cyclase activity exhibited by cyr-1 deletion mutants expressing eESAGc than was seen in yeast expressing the endogenous CYR gene from plasmids.
In addition to ESAG4, cloned from the VSG region of Trypanosoma brucei, at least three other genes cloned from T. brucei, GRESAG 4.1, 4.2 and 4.3, bear sequence homology to eukaryotic adenylate and guanylate cyclases (Alexandre et al. (1990) Mol. Biochem. Parasitol. 43, 279-288). ["GRESAG" indicates Genes Related to Expression Site Associated Genes.] It has been demonstrated that both ESAG 4 and GRESAG 4.1 can complement a S. cerevisiae adenylyl cyclase deletion mutant, cyr1.DELTA.. The trypanosome cyclases associate with the yeast membrane fraction, differ in their response to Ca.sup.2+, and do not appear to be properly regulated in yeast [Paindavoine et al. (1992) Mol. Cell. Biol. 12, 1218-1225].
Thus, the heterogenous adenylyl cyclases that have been shown to exhibit activity, although unregulated, in yeast are derived from trypanosome species. The trypanosome cyclase genes lie in regions near sequences encoding leucine-rich motifs with homology to a regulatory domain of yeast adenylyl cyclase. This suggests that proteins which derive from the two different trypanosome sequences may interact to form a regulatory complex. This could be analogous to the situation in Saccharomyces cerevisiae where activity of adenylyl cyclase is controlled through the interaction of the enzyme with regulatory RAS proteins. The homologies of sequence and regulation between the yeast and trypanosome enzymes appear to have favored the complementation of yeast deleted for adenylyl cyclase with sequences encoding the trypanosome enzyme.
Attempts to Express Mammalian Adenylyl Cyclases in Yeast
Previous attempts by other laboratories to express mammalian adenylyl cyclase in yeast were unsuccessful. Ronald Taussig, working in the laboratory of Alfred Gilman at the University of Texas Southwestern Medical Center, attempted to express mammalian type 1 adenylyl cyclase in Saccharomyces cerevisiae (personal communication). The protocol used by Taussig involved rescue of cyc cells by transformation with mammalian type 1 adenylyl cyclase; the metric of cyclase activity was growth of the test cells on forskolin-containing medium. Forskolin is known to bind directly to and to stimulate adenylyl cyclase types 1-6 in mammalian cells. Taussig was unable to detect enzyme activity in cyc cells transformed with the mammalian enzyme, i.e., he was unable to detect growth of transformed cells on forskolin-containing medium.
Expression of Mammalian Adenylyl Cyclases in Dictyostelium Discoideum.
The mammalian type 2 cyclase has been functionally expressed, by means not publicly disclosed, in the primitive eukaryote Dictyostelium discoideum [personal communication from P. Devreotes cited in Iyengar (1993)]. The structure of one of the two adenylyl cyclase genes that have been isolated from Dictyostelium, ACA, is predicted to be structurally analogous to the mammalian cyclases in that it is also an integral membrane protein [Pitt et al. 1992]. In addition, Dictyostelium can express eight G.alpha. subunits, each bearing approximately 45% sequence homology to mammalian G.alpha. proteins [Hadwiger et al. 1991; Wu and Devreotes 1991]. The lack of success in Gilman's laboratory at expressing a functional mammalian type 1 adenylyl cyclase in yeast, and the successful expression of the mammalian enzyme in Dictyostelium, indicate that differences in the transduction of signal to this enzyme exist between yeast and the higher eukaryotes. Furthermore, those differences must be taken into consideration in any attempt to recapitulate a signal transduction pathway with mammalian adenylyl cyclase in yeast.
Peptide Libraries
Peptide libraries are systems which simultaneously display, in a form which permits interaction with a target, a highly diverse and numerous collection of peptides. These peptides may be presented in solution (Houghten 1991), or on beads (Lam 1991), chips (Fodor 1991), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull 1992) or on phage (Scott, Devlin, Cwirla, Felici, Ladner '409). Many of these systems are limited in terms of the maximum length of the peptide or the composition of the peptide (e.g., Cys excluded). Steric factors, such as the proximity of a support, may interfere with binding. Usually, the screening is for binding in vitro to an artificially presented target, not for activation or inhibition of a cellular signal transduction pathway in a living cell. While a cell surface receptor may be used as a target, the screening will not reveal whether the binding of the peptide caused an allosteric change in the conformation of the receptor.
Ladner, U.S. Pat. No. 5,096,815 describes a method of identifying novel proteins or polypeptides with a desired DNA binding activity. Semi-random ("variegated") DNA encoding a large number of different potential binding proteins is introduced, in expressible form, into suitable host cells. The target DNA sequence is incorporated into a genetically engineered operon such that the binding of the protein or polypeptide will prevent expression of a gene product that is deleterious to the cell under selective conditions. Cells which survive the selective conditions are thus cells which express a protein which binds the target DNA. While it is taught that yeast cells may be used for testing, bacterial cells are preferred. The interactions between the protein and the target DNA occur only in the cell, not in the periplasm, and the target is a nucleic acid, not a protein.
Substitution of random peptide sequences for functional domains in cellular proteins permits some determination of the specific sequence requirements for the accomplishment of function. Though the details of the recognition phenomena which operate in the localization of proteins within cells remain largely unknown, the constraints on sequence variation of mitochondrial targeting sequences and protein secretion signal sequences have been elucidated using random peptides (Lemire et al., J. Biol. Chem. 264, 20206 (1989) and Kaiser et al. Science 235, 312 (1987), respectively).
Yeast have been engineered to express foreign polypeptide variants to be tested as potential antagonists of mammalian receptors. Libraries encoding mutant glucagon molecules were generated through random misincorporation of nucleotides during synthesis of oligonucleotides containing the coding sequence of mammalian glucagon. These libraries were expressed in yeast and culture broths from transformed cells were used in testing for. antagonist activity on glucagon receptors present in rat hepatocyte membranes (Smith et al. 1993).
All references cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art.